Identification of Two Linear Epitopes on MGF_110-13L Protein of African Swine Fever Virus with Monoclonal Antibodies

Abstract

African swine fever caused by African swine fever virus (ASFV) is an acute, highly contagious swine disease with high mortality. To facilitate effective vaccine development and find more serodiagnostic targets, fully exploring the ASFV antigenic proteins is urgently needed. In this study, the MGF_110-13L was identified as an immunodominant antigen among the seven transmembrane proteins. The main outer-membrane domain of MGF_110-13L was expressed and purified. Two monoclonal antibodies (mAbs; 8C3, and 10E4) against MGF_110-13L were generated. The epitopes of two mAbs were preliminary mapped with the peptide fusion proteins after probing with mAbs by enzyme-linked immunosorbent assay (ELISA) and Western blot. And the two target epitopes were fine-mapped using further truncated peptide fusion protein strategy. Finally, the core sequences of mAbs 8C3 and 10E4 were identified as ⁴⁸WDCQDGICKNKITESRFIDS⁶⁷, and ¹²²GDHQQLSIKQ¹³¹, respectively. The peptides of epitopes were synthesized and probed with ASFV antibody positive pig sera by a dot blot assay, and the results showed that epitope 10E4 was an antigenic epitope. The epitope 10E4 peptide was further evaluated as a potential antigen for detecting ASFV antibodies. To our knowledge, this is the first report of antigenic epitope information on the antigenic MGF_110-13L protein of ASFV.

Keywords: African swine fever virus; MGF_110-13L protein; epitope; monoclonal antibody.

Figure 1

Identification of antigenic transmembrane proteins with pig anti-ASFV serum. Seven predicted ASFV transmembrane protein outer-membrane domain-expressing plasmids were transiently transfected into the HEK-293 cells. The protein expression was verified with 6× His tag specific antibody by Western blot (A). After verification, transiently expressed proteins were probed with pig anti-ASFV serum from pigs vaccinated with attenuated ASFV and challenged with a virulent ASFV strain (B).

Figure 2

Expression of MGF_110-13L protein and generation of mAbs against the MGF_110-13L protein of ASFV. After transfection and selection, a stable cell line expressing the MGF_110-13L protein was established. The cell line was identified by IFA with 6× His tag specific antibody (A). The recombinant protein was purified by nickel affinity chromatography from the supernatants of the cell line and analyzed by SDS-PAGE and Western blot (B). The purified recombinant protein was treated with PNGase F and analyzed by SDS-PAGE (C). The purified proteins were analyzed by SDS-PAGE under non-reducing conditions (without β-mercaptoethanol) and reducing conditions (with β-mercaptoethanol) (D). The titers of MGF_110-13L-specific antibody in immunized pig sera were detected by ELISA (E). The titrations of monoclonal antibodies 8C3 and 10E4 were detected by ELISA (F). The MGF_110-13L proteins in ASFV-infected cell lysates were detected by Western blot with mAbs 8C3 and 10E4, respectively (G). **, glycosylated monomer. *, unglycosylated monomer.

Figure 3

Expression of peptide fusion proteins and mapping the epitopes of mAbs. (A) Diagrammatic representation of the truncated overlapping short peptides that span the outer-membrane domain of the MGF_110-13L protein. (B) SDS-PAGE analysis of recombinant peptide fusion proteins. ELISA analysis results show that peptides P3 and P4 were recognized by mAb 8C3 (C), and peptide P8 was recognized by mAb 10E4 (D). Western blot results showed that mAb 8C3 recognized peptides P3 and P4 (E) and mAb 10E4 recognized peptide P8 (F).

Figure 4

Fine-mapping of the epitope of mAb 8C3. Peptide P4 was truncated sequentially at the carboxyl or amino terminus, as shown in the schematic diagram (A). The MBP-peptide fusion proteins were expressed and analyzed by SDS-PAGE (B). The MBP-peptide fusion proteins were probed with mAb 8C3 using Western blot (C) and ELISA (D). The core sequence of the epitope of mAb 8C3 was deduced to be ⁴⁸WDCQDGICKNKITESRFIDS⁶⁷. The core sequences of the epitope are highlighted in red, and the truncated epitope sequences with reduced binding capacity to the mAb are shown in orange or yellow (A).

Figure 5

Fine-mapping of the epitope of mAb 10E4. Peptide P8 was truncated sequentially at the carboxyl or amino terminus, as shown in the schematic diagram (A). The MBP-peptide fusion proteins were expressed and analyzed by SDS-PAGE (B). The MBP-peptide fusion proteins were probed with mAb 10E4 using Western blot (C) and ELISA (D). The core sequence of the epitope of mAb 10E4 was deduced to be ¹²²GDHQQLSIKQ¹³¹. The core sequences of the epitope are highlighted in red, and the truncated epitope sequences with reduced binding capacity to the mAb are shown in orange (A).

Figure 6

Detection of African swine fever virus antibodies with synthesized peptides. The synthesized peptides EP4 of the mAb 8C3 epitope and EP8 of the mAb 10E4 epitope were subjected to detection of epitope specific antibodies in ASFV-infected pig sera using the dot blot assay (A). The synthesized peptide EP8 of the mAb 10E4 epitope was further evaluated in detecting ASFV antibodies with attenuated ASFV-infected pig sera (B) and naturally ASFV-infected pig sera (C) by dot blot assay.